Our laboratory is focused on determining the pathogenesis mechanism(s) of age related macular degeneration (AMD). AMD is a complex disease that involves the aging process but is mediated by multiple genetic and environmental factors. The choriocapillaris and Bruch's membrane are known to gradually accumulate LDL-cholesterol as a process of aging. Since these are the tissues most likely involved in the pathogenesis of AMD, we are studying the possible biochemical implications of this age-related accumulation. Our hypothesis is that these LDL deposits are likely to interfere with the permeability of Bruch's membrane and their oxidation could expose the RPE, with its numerous lipoprotein receptors, to the cytotoxic effect of the oxidized cholesterols. The internalization of the oxidized LDL by the RPE may induce a gradual loss of function and cytotoxicity. Another significant problem associated with the accumulation of LDL is the involvement of the immune system. The combination of the RPE cytotoxicity and immune system involvement may initiate the early symptoms of AMD (e.g. drusen deposits, loss of pigmentation). Once these processes begin they may eventually involve other pathways leading to choroidal neovacularization and photoreceptor degeneration. Although the age-related factors may initiate the pathogenesis of AMD, the severity and age of onset are likely due to subtle genetic and/or environmental factors. Oxysterols are known to induce potent pharmacological effects on different cell types and we are interested in understanding the cytotoxicity mechanism in RPE cells. We have tested several different oxysterols naturally found in oxidized LDL and found them to induce cell death in the cultured RPE cells. We find that cultured RPE cells are also highly susceptible to oxidized LDL. Although apoptosis has been reported as the mode of cell death in oxysterol cytotoxicity, all of our analyses thus far suggest that the death of the cultured RPE cells is unrelated to apoptosis. In order to better understand the mechanism(s) of oxysterol action we are studying proteins that bind oxysterols. We have discovered and characterized a family of 12 different oxysterol binding proteins (OSBPs) that are highly conserved and have different tissue expression and oxysterol binding characteristics. The OSBPs in general have two distinct domains, the oxysterol binding domain towards the C-terminus and a pleckstrin homology domain (PH) towards the N-terminus. The PH domains are known to interact with phosphoinositols and other membrane components and seem to target proteins to different cellular organelles. We are in the process of studying their intracellular targeting using GFP fusion constructs containing whole or portions of several selected OSBPs. We have constructed several RPE cell lines overexpressing some of the OSBPs and have determined that this overexpression protects the cells from oxysterol cytotoxicity. Using rats we are examining how LDL enters the retina and accumulates in Bruch's membrane in vivo. We have also found that LDL is capable of delivering hydrophobic molecules to the retina and RPE.